Implementation of universal newborn bloodspot screening for sickle cell disease and other clinically significant haemoglobinopathies in England: screening results for 2005-7.

Early results from the National Health Service Sickle Cell and Thalassaemia Screening programme covering the whole of England are reported following the implementation of the national newborn blood-spot screening programme. Of the 13 laboratories performing screening, 10 chose high-performance liquid chromatography as the first screen, with isoelectric focusing as the second confirmatory test. Screening results for April 2005 to March 2007 are presented and include data from all the laboratories screening newborns in England, and almost 1.2 million infants. The screen-positive results show a national birth prevalence of almost 1 in 2000. The birth prevalence in London is five times that of most of the rest of the country. Over 17,000 carriers have been identified. Approximately seven per 1000 samples are reported as post-transfusion with wide ethnic category variation. Given the prevalence of the conditions, and coverage by ethnicity, 3-4 screen-positive cases could be missed each year. National implementation of newborn screening in England has increased the number of children identified with sickle cell disease, in many areas almost doubling the workload. Underascertainment of the condition has allowed a downplaying of the scale of need. It may also have contributed to infant mortality rates in urban areas as babies died without a diagnosis or treatment. The value of a co-ordinated national approach to policy development and implementation is emphasised by the English experience. The programme provides a model for Europe as well as other countries with significant minority populations, such as Canada. Potentially it also offers important lessons for Africa where the World Health Organization is supporting the introduction of newborn screening.

Implications of increased haemoglobin A2 values in HIV positive women in the antenatal clinic.

Universal antenatal haemoglobinopathy screening in this hospital has identified several women with increased haemoglobin A(2) values, but without hypochromic microcytic red cell indices. This report describes two cases where there is evidence that the raised haemoglobin A(2) value is not caused by heterozygous beta thalassaemia, but rather results from these patients being human immunodeficiency virus (HIV) positive and on antiretroviral therapy. This will have important implications as universal antenatal haemoglobinopathy screening becomes more widespread, and as the number of HIV positive women of childbearing age increases.

Neonatal screening for haemoglobinopathies: the results of a 10-year programme in an English Health Region.

Neonatal identification of sickle cell disease can significantly reduce mortality and morbidity during the first 5 years of life. During a 10-year period, 414,801 neonates were screened by isoelectric focusing. The most common variants detected were haemoglobins S, C, D and E. Two hundred and fifty of the samples tested were homozygotes or compound heterozygotes, and 6554 samples were heterozygotes for the common variants. The gene frequencies in the population tested were calculated from this data for the most common variants. They were: S, 0.0057; C, 0.0014; D(Punjab(Los Angeles)), 0.0007; E, 0.0005. Additionally, 16 babies had beta thalassaemia major and 405 had rarer variants, of which six had never previously been described. Knowledge of the distribution of these inherited diseases is useful in healthcare planning and appropriate allocation of resources, while counselling targeted at appropriate couples enables informed parental choice and may prevent disease.

Different patterns of DNA fragmentation and degradation of nuclear matrix proteins during apoptosis induced by radiation, hyperthermia or etoposide.

Several nuclear matrix proteins are substrates for proteolytic cleavage during apoptosis. Using Western blotting, the temporal patterns of cleavage of three nuclear matrix proteins (lamin B, NUMA and the nucleoporin TPR) were compared in HL60 cells induced to undergo apoptosis after irradiation, heat shock or treatment with etoposide. Flow cytometry was used to compare the kinetics of post-cleavage degradation of lamin B, NUMA and TPR after irradiation, and to correlate DNA fragmentation with protein degradation in cells induced to undergo apoptosis with different agents. During radiation-induced apoptosis, cleavage and subsequent degradation of lamin B, NUMA and TPR occurred with different kinetics. Low-molecular-weight DNA fragmentation occurred subsequent to the initiation of NUMA cleavage, coincided with lamin B cleavage, but occurred before more extensive degradation of lamin B and NUMA. A similar sequence was observed for cells treated with etoposide. However, during heat-induced apoptosis, cleavage of lamin B and NUMA occurred much sooner compared to other agents, with NUMA cleaved into multiple fragments within 15 min after heating. We conclude that the hierarchical sequence and kinetics of degradative events contributing to nuclear disassembly during apoptosis are highly dependent on the inducing agent. Furthermore, the nuclear pore complex, like the nuclear lamina and internal nuclear matrix, is a target for proteolytic cleavage.

Two new variants with the same substitution at position beta122: Hb Bushey [beta122(GH5)Phe-->Leu] and Hb Casablanca [beta65(E9)lys-->Met; beta122(GH5)Phe-->Leu].

Hb Bushey, found in a Chinese baby and his father, is a new variant with a point mutation leading to the substitution Phe-->Leu at position beta122. Hb Casablanca, found in a family from Morocco, is a further example of a hemoglobin variant that carries two abnormalities in the same chain; the first is identical to that of Hb Bushey and the second to that of Hb J-Antakya [beta65 (E9)Lys-->Met]. Structural abnormalities of both Hbs were determined by protein chemistry methods including electrospray and tandem mass spectrometry. Their stability and oxygen binding properties were found to be identical to those of Hb A. Various mechanisms that may lead to two point mutations in the same chain are reviewed briefly.

Organisation and cost-effectiveness of antenatal haemoglobinopathy screening and follow up in a community-based programme.

OBJECTIVE: To consider the organisation cost and effectiveness, of universal, community-based antenatal screening for the haemoglobinopathies, and to estimate the cost-effectiveness of programmes at different levels of prevalence and mix of haemoglobinopathy traits. DESIGN: Retrospective review of laboratory and Sickle Cell and Thalassaemia Centre worksheets with costing of capital equipment, consumables, salaries and overheads, and estimation of costs in a range of circumstances. SETTING: A haematology department, including a Sickle Cell and Thalassaemia Centre, providing antenatal and neonatal screening programmes in Inner London. PARTICIPANTS: Two thousand one hundred and one women booking at the antenatal clinic whose samples were referred for screening during 1994. MAIN OUTCOME MEASURES AND RESULTS: In addition to assessing the cost-effectiveness of antenatal haemoglobinopathy screening in a number of settings, the following specific financial information was assembled for the service in Brent: 1. cost of identifying abnormal haemoglobin in mother (l209); 2. cost of identifying at-risk fetus before confirmation by prenatal diagnosis (l2,455); 3. cost of providing genetic information and counselling to mother with abnormal haemoglobin (l109); 4. programme savings from cases averted (l61,000). Conclusions Antenatal screening with follow up counselling can be self-financing at most prevalences of haemoglobinopathy traits, with greater savings where a high proportion of the traits are beta thalassaemia. There is a net financial cost (l1,350) only at prevalences below 2.5% of traits if these are mainly for sickle cell disease. Since there are other benefits is it likely that antenatal screening will be considered cost-effective even at quite low levels of trait prevalence.

Degradation of the nuclear matrix is a common element during radiation-induced apoptosis and necrosis.

Human promyelocytic leukemia (HL60) cells were irradiated with 10 or 50 Gy of X rays and studied for up to 72 h postirradiation to determine the mode of death and assess changes in the nuclear matrix. After 50 Gy irradiation, cells were found to die early, primarily by apoptosis, while cells irradiated with 10 Gy died predominantly by necrosis. Disassembly of the nuclear lamina and degradation of the nuclear matrix protein lamin B occurred in cells undergoing radiation-induced apoptosis or necrosis. However, using Western blotting and a recently developed flow cytometry assay to detect changes in nuclear matrix protein content, we found that the kinetics and mechanisms of disassembly of the nuclear lamina are different for each mode of cell death. During radiation-induced apoptosis, cleavage and degradation of lamin B to a approximately 28-kDa fragment was detected in most cells within 4-12 h after irradiation. Measurements of dual-labeled apoptotic cells revealed that nonrandom DNA fragmentation was evident prior to or concomitant with breakdown of the nuclear lamina. Disassembly of the nuclear lamina during radiation-induced necrosis occurred much later (between 30-60 h after irradiation), and a different cleavage pattern of lamin B was observed. Degradation of the nuclear lamina was also inhibited in apoptosis-resistant BCL2-overexpressing HL60 cells exposed to 50 Gy until approximately 48 h after irradiation. These data indicate that breakdown of the nuclear matrix may be a common element in radiation-induced apoptosis and necrosis, but that the mechanisms and temporal patterns of breakdown of the nuclear lamina during apoptosis are distinct from those of necrosis.

Evaluation of cation-exchange HPLC compared with isoelectric focusing for neonatal hemoglobinopathy screening.

BACKGROUND: Central Middlesex Hospital, in northwest London, has screened neonates for hemoglobinopathies, using the established manual technique of isoelectric focusing (IEF) since 1989. Recently, this laboratory has faced a large increase in the number of samples tested per year. This study compared the detection of hemoglobin abnormalities between the existing manual IEF method and that of automated cation-exchange HPLC to determine the reliability of HPLC and whether an automated system would save time in the laboratory. METHODS: Over a 15-month period, 25 750 blood samples, collected by heel prick onto filter paper, were tested using HPLC, and the results were compared with those obtained with IEF. RESULTS: HPLC and IEF each identified 568 patients with FAS, 151 with FAC, 49 with FAD-Punjab, 23 with FS, 3 with FC, 6 with FSC, 5 with FE, and 1 with FD. IEF detected 62 patients with FAE, whereas HPLC detected 63. This additional FAE was observed on repeat IEF. One additional heterozygote detected by HPLC was initially not observed by IEF, but was detected on repeat IEF. HPLC detected all but six cases of Hb Barts observed by IEF. One double heterozygote and four heterozygotes were detected by IEF, but not by HPLC. The detection of hemoglobin variants expressed at low concentrations was comparable for the two methods, and carryover was not observed in routine analysis on HPLC. CONCLUSIONS: HPLC is a sensitive, efficient, and time-saving alternative to IEF for the neonatal screening of common hemoglobinopathies.

Costing model for neonatal screening and diagnosis of haemoglobinopathies.

AIM: To compare the costs and cost effectiveness of universal and targeted screening for the haemoglobinopathies; to compare the cost of two laboratory methods; and to estimate the cost effectiveness of programmes at different levels of prevalence and mix of haemoglobinopathy traits. METHODS: A retrospective review of laboratory and follow up records to establish workload and costs, and estimation of costs in a range of circumstances was made in a haematology department and sickle cell and thalassaemia centre, providing antenatal and neonatal screening programmes in Inner London. The costs for 47,948 babies, screened during 1994, of whom 25 had clinically significant haemoglobinopathies and 704 had haemoglobinopathy traits, were retrospectively assessed. RESULTS: The average cost per baby tested (isoelectric focusing and high power liquid chromatography) was 3.51 Pounds /3.83 Pounds respectively; the cost per case of sickle cell disease identified (IEF/HPLC) was 6738 Pounds /7355 Pounds; the cost per trait identified (IEF/HPLC) was 234 Pounds /255 Pounds; the cost per extra case of SCD and trait identified by universal programme varied. CONCLUSIONS: IEF and HPLC are very similar in terms of average cost per test. At 16 traits/1000 and 0.5 SCD/1000 there was no significant identification cost difference between universal and targeted programmes. Below this prevalence, a targeted programme is cheaper but likely to miss cases of SCD. If targeted programmes were 90-99% effective, universal programmes would cease to be good value except at very high prevalence. Greater use of prenatal diagnosis, resulting in termination, and therefore fewer affected births, reduces the cost effectiveness of universal screening. Screening services should aim to cover a screened population which will generate a workload over 25,000 births a year, and preferably over 40,000.

Alteration of an intersubunit contact in hemoglobin variants: comparative study of modifications at position alpha 126 Asp (H9).

Four human hemoglobin variants have already been described at position alpha 126 (H9), which is normally occupied by an aspartate: Hb Montefiore (-->Tyr), Hb Tarrant (-->Asn), Hb Fukutomi (-->Val), Hb Sassari (-->His). An additional variant, Hb West One (alpha 126 (H9) Asp-->Gly) is herein described. Aspartate alpha 126 (H9) is involved in a set of hydrogen bonds and salt bridges located at the C-terminal portion of the alpha-chains and of the C-helix of the beta-chains, which are broken in the oxy conformer, providing one of the most important sources of the difference in free energy between the T- and R-state in hemoglobin. A comparative study of four of these alpha 126 Hb variants is presented. An identical degree of alteration of the oxygen binding properties (increased oxygen affinity and decreased cooperativity) was found in all cases, when measured under standard experimental conditions (pH 7.2, 0.1 M NaCl). In contrast, the effect of L345 (a derivative of bezafibrate, which is a specific alpha-chain binding effector) on oxygen binding to Hb differed from one variant to another. When a bulky Tyr or His residue occupied the alpha 126 (H9) position, little effect of L345 was observed. Conversely, when this position was occupied by a residue of smaller size (Gly or Asn), normal heterotropic effects were observed. Molecular graphic modelling indicates that two classes of three-dimensional structure modifications may occur.

Hb Uxbridge [beta 20 (B2)Val-->Gly]: a new variant with mild increase in oxygen affinity found during a neonatal screening program.

Hb Uxbridge [beta 20(B2)Val-->Gly] was found in an English family during a neonatal hemoglobinopathy screening program. In both the child and the parent carrying this hemoglobin variant, the red cell parameters were normal. By isoelectrofocusing Hb Uxbridge appeared to have an isoelectric point slightly higher than Hb A but was silent on cellulose acetate and acid gel electrophoresis. Structural modifications affecting position beta 20(B2) have been demonstrated to be responsible for a high oxygen affinity and polycythemia in Hb Olympia (Val-->Met) and Hb Trölhattan (Val-->Glu). In the case of Hb Uxbridge, despite an alteration of the same site, the oxygen binding parameters of the patient's hemolysate showed only a mild (ca. 20%) increase in oxygen affinity.

Flow cytometric analysis of nuclear matrix proteins: method and potential applications.

The nuclear matrix (NM) is an important structural component of the nucleus that participates in the regulation of several diverse metabolic processes. Immunometric assays have shown that alterations in NM-associated functions and morphological characteristics may occur as a result of changes in NM composition. Recent evidence suggests that detection of quantitative or qualitative changes in nuclear matrix protein (NMP) composition may be useful in the diagnosis of cancer and as a reliable indicator of cell death. We have developed an in situ flow cytometric technique for the simultaneous detection of specific NMPs and DNA content in fixed, permeabilized cells. Illustrative results from two different applications of these methods involving two different cell lines (human melanoma and promyelocytic leukemia) are presented, including: 1) measurements of NM breakdown in necrotic and apoptotic cells after treatment with the cytotoxic agents camptothecin, etoposide, or hyperthermia; and 2) detection of changes in NMP content immediately after heat shock. We demonstrate that the technique is useful for the identification of cell-cycle specificity of NM breakdown and allows correlations to be made between the kinetics of DNA fragmentation and NMP solubilization. Furthermore, our studies indicate that flow cytometric detection of changes in NM composition may be useful for identifying different modes and temporal patterns of cell death. We discuss other potential applications of the technique and advantages over standard biochemical assays.

Leukemia inhibitory factor and interleukin-11 promote maturation of murine and human megakaryocytes in vitro.

The growth-promoting activities of optimally stimulating concentrations of leukemia inhibitory factor (LIF) and interleukin-11 (IL-11), a stromal cell-derived cytokine, on megakaryocytes in liquid marrow cultures were compared to interleukin-6 (IL-6), a known megakaryocytes maturation factor. Maximally stimulating concentrations of LIF (25 ng/ml), IL-11 (10 ng/ml), or IL-6 alone (10 ng/ml) promoted an 81, 157, and 153% increase, respectively, in acetylcholinesterase (AchE) activity in murine serum-free cultures compared with controls (n = 5). In combination with 25 U/ml murine interleukin-3 (IL-3), LIF, IL-6, and IL-11 showed increases, respectively, of 35%, 49%, and 174% in AchE activity compared with IL-3 alone (n = 4). Flow cytometric analysis of 4-day-old cultures showed that LIF alone had minimal effect on megakaryocytic ploidy, whereas IL-11 and IL-6 alone markedly augmented high ploidy cells. Enumeration of cells stained for AchE showed that IL-11 increased the numbers of Mks in comparison to LIF, IL-6 or controls by up to 59%. Moreover, a twofold increment in Mk number was noted when IL-11 was used in combination with IL-3 (compared with either IL-3 alone of IL-3+IL-6). Flow cytometric ploidy analysis of 8-day-old human liquid marrow cultures showed that either LIF, IL-11, or IL-6 alone markedly augmented the percentage of 32N cells compared with cultures containing only human IL-3. The data suggest that LIF and IL-11 promote murine and human Mk maturation in vitro, although the effect of IL-11 exceeds that of LIF in mice. Despite the comparable influence of IL-11 and IL-6 on Mk ploidy, IL-11 has the additional characteristic of enhancing the number of Mks, particularly in combination with IL-3.

Serum interleukin-6 levels in patients with thrombocytosis.

Interleukin-6 (IL-6) has been shown to increase platelet counts in several animal models and to enhance megakaryocytopoiesis in vitro. In order to investigate the possible relationship between IL-6 and thrombocytosis, serum IL-6 levels in patients with platelet counts > or = 6 x 10(5)/microliters were measured using an IL-6-responsive bioassay. A cohort of healthy volunteers with normal platelet counts was used to establish a control mean serum IL-6 level [2.19 U/ml +/- 1.08 SD (range 0-5.5)]. Patients with primary thrombocytosis had a mean serum IL-6 level not significantly different from controls. In comparison, serum IL-6 levels of patients with reactive thrombocytosis were significantly greater than controls (38.3 U/ml +/- 94.6; range 0-933; P < 0.001). Although no significant correlation was observed between the degree of serum IL-6 elevation and the height of the platelet count in any individual, elevated serum IL-6 was highly correlated with reactive thrombocytosis.

Thrombocytopoiesis in normal and sublethally irradiated dogs: response to human interleukin-6.

The response of megakaryocytes and platelets to the administration of recombinant human interleukin-6 (IL-6) was investigated in normal and sublethally irradiated dogs. IL-6 was administered for 2 weeks at doses of 10 to 160 micrograms/kg/d to normal animals to assess dose-response and toxicity. Subsequently, 40, 80, or 160 micrograms/kg/d for 2 weeks was administered to animals treated with 200 cG total body irradiation. Analysis of normal dogs showed a significant increment in the platelet count detectable approximately 11 days after initiation of IL-6 at all administered doses. Large platelets greater than 6.3 microns in diameter were observed 1 day after beginning IL-6, progressively increasing to as many as 19.1% of the total circulating platelets by day 10. The ploidy distribution of the marrow megakaryocytes did not differ from the normal at doses of less than or equal to 80 micrograms/kg/d, but at 160 micrograms/kg/d, a shift toward higher ploidy cells was noted. No change in total white count was noted; however, a decrease in hematocrit was seen at all doses. In the irradiated animals, the platelet count recovered earlier in the IL-6-treated dogs than in the controls, but no consistent change in the ploidy distribution was observed irrespective of dose. Large platelets were also noted in the treated animals, comprising up to 6.9% of the total platelet count. Fibrinogen levels were elevated to greater than 4 times normal. A significant decrease in hematocrit was seen in all animals, while no consistent change was noted in the white count. Elevations in serum cholesterol, triglycerides, and alkaline phosphatase, together with a decline in serum albumin were observed in all the treated animals (both normal and irradiated), but clinical symptoms were observed only in the dogs receiving greater than or equal to 80 micrograms/kg/d. The data show that IL-6 alone is capable of enhancing platelet recovery in dogs with bone marrow suppression.

Regulation of interleukin 6 expression in murine bone marrow stromal cells.

The regulation of interleukin 6 (IL-6) expression in the B-lymphocyte-supporting murine stromal cell line BMS2 has been examined in response to exogenous cytokines and chemical agents. Kinetic analyses of IL-6 mRNA induction and decay are presented together with analysis of the IL-6 biological activity. The cytokines tumor necrosis factor, interleukin 1 (alpha and beta), and transforming growth factor beta, as well as forskolin and dibutyryl cyclic AMP, all induce a transient rise in the steady-state level of IL-6 mRNA and an increased release of IL-6 protein. To study its regulation at the chromatin level, the murine IL-6 genomic gene has been cloned. Induction of IL-6 expression correlates with increased DNA nicking, consistent with increased topoisomerase I and endogenous nuclease activity. This finding is supported by kinetic analyses using camptothecin, a topoisomerase I inhibitor. We conclude that IL-6 regulation in murine stromal cells capable of supporting B-lymphopoiesis is comparable to that observed in human diploid fibroblasts.

Elevated serum interleukin-6 levels in patients with reactive thrombocytosis.

Interleukin-6 (IL-6) is known to promote megakaryocytopoiesis in vitro and raise platelet counts in vivo. To determine if there is a relationship between circulating IL-6 and thrombocytosis in man, we measured bioactive IL-6 in the serum of 13 patients with myeloproliferative disorders and 143 patients with reactive thrombocytosis having platelet counts greater than or equal to 600 x 10(9)/l. IL-6 activity was assayed using the IL-6-responsive B9 cell line. Seventy-one controls with normal platelet counts had a mean IL-6 level of 2.19 U/ml +/- 1.08 (SD). None of the 13 patients with myeloproliferative disorders had elevated IL-6 levels (1.56 U/ml +/- 1.2). In contrast, serum IL-6 levels of 143 patients (158 samples) with reactive thrombocytosis were significantly greater than controls (38.3 U/ml +/- 94.6; P less than 0.001), with 83% of the samples showing elevated serum IL-6. No significant correlation was observed between serum IL-6 levels and platelet counts in the reactive thrombocytosis group. We conclude that elevated IL-6 is associated with reactive thrombocytosis, and hypothesize that the increased platelet count in many cases is causally related to elevated IL-6.